Activation of T-cells in response to antigen-presenting stimuli is an initial event in the immune response. The plasma membrane T-cell receptor binds the antigen-presenting stimulus and a cascade of signalling responses culminating in lymphocyte proliferation and differentiation ensues. T-cell receptor signal transduction parallels the mitogenic response pathways characterized in fibroblasts; however the T-cell signalling pathway appears to be more complex and to involve intermediate tyrosine kinases and serine/threonine kinases which are unique to the immune system. Src family kinases (p56lCk, p59fyn and ZAP7O are activated by T-cell receptor engagement, and activation of a Ras/Raf-mediated MAPK pathway has been described. More recently, activation of a second pathway, perhaps through the tyrosine kinase Tec , has been described. This pathway is activated through the CD28 receptor and results in phosphorylation and activation of the transcription factor c-Jun by the protein kinase JNK. At this point the relative roles and importance of these signalling pathways remain unresolved. This project intends to investigate the role of the serine/threonine kinase S6/H4 kinase in Jurkat (T-lymphoblasts) activation. S6/H4 kinase is the mammalian homologue of STEE2O and is implicated in receptor-mediated responses involving reorganization of cytoskeletal elements, including membrane ruffling. Autophosphorylation is required for enzyme activation, but upstream signals and downstream substrates which mediate this activation have not been elucidated. The long term goals of this research are to establish the signalling pathway in which S6/H4 kinase participates and to elucidate the role of this kinase in T cell activation. Specific aims which will be pursued in this phase of the work are as follows: 1) determination of the phosphorylation sites which occur in S6/H4 kinase in vivo; 2) determination of the-effect of tyrosine kinase activation on serine, threonine, and tyrosine phosphorylation and activation of S6/H4 kinase in vivo; 3) investigation of cellular substrates of S6/H4 kinase with particular emphasis on API (Jun/Fos) and the myosin light chain kinase, 4) effects of transfection with constituitively active and dominant negative S6/H4 kinase constructs on signalling in T cells. Results from these experiments will provide initial insights into the role of the S6/H4 kinase in T cell activation. T-cell activation, or lack thereof, is central to the normal immune response and plays roles in a variety of pathologies including HIV infection, immunological anergy and proliferative disorders. Dissection of the molecular events which mediate normal proliferative and apoptic responses will increase opportunities for therapeutic intervention of T- cell disorders.